Bottom Line:
EPS2 was found to adopt a random coil structural conformation.Deletion of the entire 14-kb eps cluster resulted in an acapsular mutant phenotype that was not able to produce either EPS-2 or EPS-1.These findings provide insights into the biosynthesis and structures of novel exopolysaccharides produced by L. johnsonii FI9785, which are likely to play an important role in biofilm formation, protection against harsh environment of the gut, and colonization of the host.

ABSTRACTExopolysaccharides were isolated and purified from Lactobacillus johnsonii FI9785, which has previously been shown to act as a competitive exclusion agent to control Clostridium perfringens in poultry. Structural analysis by NMR spectroscopy revealed that L. johnsonii FI9785 can produce two types of exopolysaccharide: EPS-1 is a branched dextran with the unusual feature that every backbone residue is substituted with a 2-linked glucose unit, and EPS-2 was shown to have a repeating unit with the following structure: -6)-α-Glcp-(1-3)-β-Glcp-(1-5)-β-Galf-(1-6)-α-Glcp-(1-4)-β-Galp-(1-4)-β-Glcp-(1-. Sites on both polysaccharides were partially occupied by substituent groups: 1-phosphoglycerol and O-acetyl groups in EPS-1 and a single O-acetyl group in EPS-2. Analysis of a deletion mutant (ΔepsE) lacking the putative priming glycosyltransferase gene located within a predicted eps gene cluster revealed that the mutant could produce EPS-1 but not EPS-2, indicating that epsE is essential for the biosynthesis of EPS-2. Atomic force microscopy confirmed the localization of galactose residues on the exterior of wild type cells and their absence in the ΔepsE mutant. EPS2 was found to adopt a random coil structural conformation. Deletion of the entire 14-kb eps cluster resulted in an acapsular mutant phenotype that was not able to produce either EPS-2 or EPS-1. Alterations in the cell surface properties of the EPS-specific mutants were demonstrated by differences in binding of an anti-wild type L. johnsonii antibody. These findings provide insights into the biosynthesis and structures of novel exopolysaccharides produced by L. johnsonii FI9785, which are likely to play an important role in biofilm formation, protection against harsh environment of the gut, and colonization of the host.

Mentions:
Probing the cell surfaces of two of the L. johnsonii strains with a d-galactose-specific lectin (PA1)-functionalized AFM tip allowed an in situ discrimination of the different EPS produced, given that EPS-2 has galactose residues that are absent in EPS-1 (Fig. 3). Fig. 7 shows comparative force-volume images of the wild type and ΔepsE mutant strains, allowing the topography of the cells to be compared with the adhesive interactions detected. The left-hand panels depict topography, and the right-hand panels depict the levels of adhesion encountered by the PA-1-functionalized AFM tip at each imaging point. A close-packed cluster of wild type cells (Fig. 7A) can be seen, and a single ΔepsE mutant cell is visualized (Fig. 7C). The adhesion maps reveal that a larger number of the pixels displayed adhesion above the base-line level (∼50 pN) for the wild type sample (Fig. 7B) than the ΔepsE mutant sample (Fig. 7D). Analysis of the adhesion data captured on the two samples allowed a quantitative comparison to be made. The modal value for both samples occurs between 50 and 55 pN (Fig. 8A). Although the base-line level of adhesion appears similar for both samples, the wild type data set has a greater proportion of adhesion events in the higher value categories than the ΔepsE data set (inset), indicating a higher degree of specific interactions.

Mentions:
Probing the cell surfaces of two of the L. johnsonii strains with a d-galactose-specific lectin (PA1)-functionalized AFM tip allowed an in situ discrimination of the different EPS produced, given that EPS-2 has galactose residues that are absent in EPS-1 (Fig. 3). Fig. 7 shows comparative force-volume images of the wild type and ΔepsE mutant strains, allowing the topography of the cells to be compared with the adhesive interactions detected. The left-hand panels depict topography, and the right-hand panels depict the levels of adhesion encountered by the PA-1-functionalized AFM tip at each imaging point. A close-packed cluster of wild type cells (Fig. 7A) can be seen, and a single ΔepsE mutant cell is visualized (Fig. 7C). The adhesion maps reveal that a larger number of the pixels displayed adhesion above the base-line level (∼50 pN) for the wild type sample (Fig. 7B) than the ΔepsE mutant sample (Fig. 7D). Analysis of the adhesion data captured on the two samples allowed a quantitative comparison to be made. The modal value for both samples occurs between 50 and 55 pN (Fig. 8A). Although the base-line level of adhesion appears similar for both samples, the wild type data set has a greater proportion of adhesion events in the higher value categories than the ΔepsE data set (inset), indicating a higher degree of specific interactions.

Bottom Line:
EPS2 was found to adopt a random coil structural conformation.Deletion of the entire 14-kb eps cluster resulted in an acapsular mutant phenotype that was not able to produce either EPS-2 or EPS-1.These findings provide insights into the biosynthesis and structures of novel exopolysaccharides produced by L. johnsonii FI9785, which are likely to play an important role in biofilm formation, protection against harsh environment of the gut, and colonization of the host.

ABSTRACTExopolysaccharides were isolated and purified from Lactobacillus johnsonii FI9785, which has previously been shown to act as a competitive exclusion agent to control Clostridium perfringens in poultry. Structural analysis by NMR spectroscopy revealed that L. johnsonii FI9785 can produce two types of exopolysaccharide: EPS-1 is a branched dextran with the unusual feature that every backbone residue is substituted with a 2-linked glucose unit, and EPS-2 was shown to have a repeating unit with the following structure: -6)-α-Glcp-(1-3)-β-Glcp-(1-5)-β-Galf-(1-6)-α-Glcp-(1-4)-β-Galp-(1-4)-β-Glcp-(1-. Sites on both polysaccharides were partially occupied by substituent groups: 1-phosphoglycerol and O-acetyl groups in EPS-1 and a single O-acetyl group in EPS-2. Analysis of a deletion mutant (ΔepsE) lacking the putative priming glycosyltransferase gene located within a predicted eps gene cluster revealed that the mutant could produce EPS-1 but not EPS-2, indicating that epsE is essential for the biosynthesis of EPS-2. Atomic force microscopy confirmed the localization of galactose residues on the exterior of wild type cells and their absence in the ΔepsE mutant. EPS2 was found to adopt a random coil structural conformation. Deletion of the entire 14-kb eps cluster resulted in an acapsular mutant phenotype that was not able to produce either EPS-2 or EPS-1. Alterations in the cell surface properties of the EPS-specific mutants were demonstrated by differences in binding of an anti-wild type L. johnsonii antibody. These findings provide insights into the biosynthesis and structures of novel exopolysaccharides produced by L. johnsonii FI9785, which are likely to play an important role in biofilm formation, protection against harsh environment of the gut, and colonization of the host.